Method of producing microbial transglutaminase

ABSTRACT

The present invention relates to a method of secretory production of transglutaminase by a microorganism.  
     The object of the present invention is to provide a method of produce a large amount of transglutaminase by causing Streptomyces bacteria to produce and secrete a large amount of transglutaminase.  
     The present invention is a method of producing a large amount of transglutaminase, comprising culturing a Streptomyces bacterium harboring an expression plasmid containing a transglutaminase gene from actynomycetes and its native (naturally occurring) promoter, causing the bacterium to secrete protransglutaminase during the initial phase to the middle phase of culturing, and obtaining mature transglutaminase (active form) by cleaving and removing the pro9-structure, for example, with proteases derived from Streptomyces.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for secretoryproduction of actynomycetes-derived transglutaminase by geneticrecombinant techniques using the host-vector system of Streptomyceslividans (also referred to as S. lividans hereinafter.Transglutaminase). Transglutaminase produced and secreted according tothe present invention is widely used in the food processing ormedicines.

[0002] Transglutaminase which is produced and secreted according to thepresent invention is an enzyme which catalyzes the acyl transferreaction of γ-calboxylamide groups located in the peptide chain ofproteins. When the enzyme is reacted with a protein, ε-(γ-Glu)-Lys crosslinking reaction, the substitution reaction from Gln to Glu bydeamination of Gln can be occurred. Transglutaminase is used forproducing gelatinized foods such as jelly, or yogurt, cheese,gelatinized cosmetics or improving the quality of meat (Publication ofunexamined Japanese patent application (referred to as JP-Kokaihereinafter) No. 1-50382). It is also used for producing materials forthermostable microcapsules or carriers for immobilized enzymes. Thustransglutaminase is an enzyme highly useful in the industry.

[0003] It is previously know that there are animal-derivedtransglutaminase and bacteria-derived transglutaminase (microbialtransglutaminase, which may be referred to as MTG herein after). Theformer is a calcium dependent enzyme and distributes in the animalorgans, skin or blood and the like. The examples are, for example, humankeratinocyte transglutaminase (M. A. Phillips et al. Proc. Natl. Acad.Sci. USA, 87, 9333 (1990)), human blood coagulation factor XIII (A.Ichinose et al., Biochemistry, 25, 6900 (1990)). Regarding to thelatter, calcium independent one is discovered from Streptoverticilliumbacteria. The examples are, for example, Streptoverticilliumgriseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp.cinnamoneum (which may be referred to as S. cinnamoneum herein after)IFO 12852 and Streptoverticillium mobaraense (which may be referred toas S. mobaraense hereinafter) IFO 13819 (JP-Kokai No. 64-27471). Peptidemapping and genetic structural analysis revealed that transglutaminasesproduced by these bacteria have no homology with the enzymes fromanimals (EP 0 481 504 A1).

[0004] Microbial transglutaminases (MTGs) had problems in the amount oryield because they are produced through purification procedures from theculture of bacteria such as above described bacteria. The production oftransglutaminase using genetic engineering techniques is also attempted.Transglutaminase proteins and genes thereof are described in, forexample, JP-Kokai No. 64-27471, Biosci. Biotech. Biochem., 58, 82-87(1994), Biosci. Biotech. Biochem., 58, 88-92 (1994), JP-Kokai No.5-199883, Biochimie, 80, 313-319 (1998), Eur. J. Biochem., 257, 570-576(1998), WO 9606931 and WO 9622366, which describe the reports for theproduction by host-vector system such as S. lividans, Aspergillus oryzaeor Escherichia coli (which may be referred to as E. coli hereinafter). Aproducing method using secretory expression in microorganisms such as E.coli or yeast (JP-Kokai No. 5-199883) and a method for producingfunctional MTG by expressing MTG as an inactive fused protein inclusionbody, resolubilizing the protein inclusion body by protein denaturingagent and then reconstituting the protein (JP-Kokai No.6-30771) arereported. However, in the secretory expression by conventionaltechniques using microorganisms, the problem of very poor expressionlevel has been indicated. For secretory production of transglutaminasein Streptomyces, there is an report which describes an example includingthe specific description about secretory accumulation (Biosci. Biotech.Biochem., 58, 82-87 (1994); JP-Kokai No.5-199883), wheretransglutaminase gene from Streptoverticillium mobaraense was introducedinto Streptoverticillium lividans as a host using genetic recombinationtechniques, however, the amount of the secretion was only about 0.1mg/l.

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide a method ofproducing a large amount of transglutaminase by secretory production oftransglutaminase in Streptomyces bacteria.

[0006] The method of the present-invention is the method of producing alarge amount or transglutaminase characterized in that a host-vectorsystem for Streptomyces is used, where an expression plasmid which canhighly express a gene in Streptomyces bacteria is constructed usingtransglutaminase gene from Streptomyces, that is, the signal peptideregion and the pro-structural region and mature structural region andthe native (natural) promoter region controlling the expression oftransglutaminase, and the expression plasmid is introduced into aStreptomyces bacterium, the bacterium is cultured, and the bacterium isdirected to secrete transglutaminase having additional pro-structuralpart (pro-transglutaminase) during the initial phase to the middle phaseof culturing and then mature (active) transglutaminase is obtained inthe late phase of culturing by cleaving and removing the pro-structure,for example, with proteases derived from Streptomyces.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1. shows the construction procedure of the expression plasmidpUJ-MTG.

DETAILED DESCRIPTION OF THE INVENTION

[0008] According to the present invention, a large amount of mature(active form) transglutaminase are obtained by using a Streptomycesbacterium as a host-vector system and generating an expression constructcontaining the gene for transglutaminase having the pro-structural part(pro-transglutaminase) linked to the down stream of the native promoterfor transglutaminase gene, and by introducing the construct into aStreptomyces bacterium, expressing it and cleaving the extracellularysecreted pro-transglutaminase, for example, with proteases produced bythe same Streptomyces bacterium.

[0009] Secretory proteins are generally known to be translated aspre-peptides or prepro-peptides and then undergo post-translationalmodifications to generate mature proteins. Namely, it is generally knownthat secretory proteins are translated as pre-peptides orprepro-peptides, and then, the signal peptides (pre-part) thereof arecleaved to convert them into mature peptides or pro-peptides and thepro-parts of the pro-peptides are cleaved to generate the maturepeptides. Transglutaminase is one of such proteins. As used herein,transglutaminase having both the signal peptide and the pro-part, namelythe primary translational product, may be referred to as“prepro-transglutaminase” and transglutaminase having the pro-part butnot the signal peptide may be referred to as “pro-transglutaminase”. Thepro-part of pro-transglutaminase may be referred to as “pro-structuralpart” or simply “pro-structure”. As used herein, the “pro-structuralpart/pro-structure” of transglutaminase and the “pro-part” of theprotein are used interchangeably. Thus, “pro-transglutaminase” may alsobe referred to as “transglutaminase with additional pro-structuralpart”.

[0010] As used herein, a protein of which pro-part is “cleaved andremoved” refer to a protein wherein one or more amino acid constitutingthe pro-part is removed by cleaving peptide bond, and it includes aprotein having the identical N-terminal region to the native matureprotein and also a protein having one or more additional amino acidderived from the pro-part at its N-terminal compared to the nativeprotein or a protein having shorter amino acid sequence than that of thenative mature protein. As used herein, “mature transglutaminase” and“active form transglutaminase” are use in the same meaning.

[0011] Generally, the genetic constructs used in the present inventionare those having suitable sequences containing promoters, nucleotidefragments encoding prepro-transglutaminase and regulatory sequencesrequired to express the intended proteins in Streptomyces bacteria, atthe appropriate location. The Vector which can be used for theconstructs are not limited and any vector can be used which can functionin Streptomyces bacteria, and the vectors may be vectors whichautonomously replicate extrachromosomally such as plasmids or may bevectors which can integrated into the bacterial chromosome. Plasmidsfrom Streptomyces are preferable, and the examples include, for example,pIJ702 (J. Gen. Microbiol., 129, 2703-2714, (1983)) and the plasmidsobtained by improving it.

[0012] The promoters which can be used in the present invention toexpress transglutaminase genes in Streptomyces bacteria are the nativepromoters of transglutaminase genes from Actinomycetes. However, in somecases the promoter region cannot be identified, because the consensussequence is not established in Actinomycetes in contrast with E.coli. Insuch cases, the genetic fragment containing 5′-upstream region largeenough to covering the structural gene for transglutaminase and thepromoter region required to regulate its expression may be used.

[0013] Transglutaminase genes which can be used in the present inventionare not particularly limited, and, for example, genes for secretorytransglutaminase derived from S. cinnamoneum IFO 12852, S. grosepcarneumIFO 12776, S. mobaraence IFO 13819 and Streptoverticillium lydicus(WO96/06931) are preferable. Transglutaminase genes from S. cinnamoneumor S. mobaraense are particularly preferable, which are used with thenative promoter of the respective transglutaminase genes.

[0014] The entire nucleotide sequence of transglutaminase gene from S.cinnamoneum IF012852 containing the 5′-upstream region, which wasidentified by the inventors of the present invention, is shown in SEQ IDNO:1, and the amino acid sequence encoded by the nucleotide sequence isshown in SEQ ID NO:2. It was assumed that from amino acid at position 1to position 32 in the amino acid sequence was the sequence for pre-part,from amino acid at position 33 to position 86 was the sequence forpro-part and from amino acid at position 87 to position 416 was thesequence for mature transglutaminase. The entire nucleotide sequence oftransglutaminase gene containing the 5′-upstream region from S.mobaraense is also shown in SEQ ID NO:3, and the amino acid sequenceencoded by the nucleotide sequence is shown in SEQ ID NO:4. From aminoacid at position 1 to position 31 in the amino acid sequence is thesequence for pre-part, from amino acid at position 32 to position 76 isthe sequence for pro-part and from amino acid at position 77 to position407 is the sequence for mature transglutaminase.

[0015] Methods for introducing the genetic constructs used in thepresent invention are not particularly limited, and the protoplastprocess (Gene, 39, 281-286 (1985); JP-Kokai No. 3-251182),electroporation process (Bio/Technology, 7, 1067-1070 (1989)) may beused. The thus obtained transformants may be cultured by usingconventional methods and conditions. The medium for culturing thesemicroorganisms may be a conventional medium, for example, a mediumcontaining carbon sources, nitrogen sources and inorganic ions. It ispreferable to add vitamins, organic micronutrients such as amino acidsor natural materials such as polypeptone or yeast extract. As carbonsources, carbohydrates such as solubilized starch, glucose or sucrose,organic acids and alcohols are properly used. The culture is conductedunder the aerobic condition for one day to 2 weeks appropriatelymaintaining pH ranging from 5.0 to 8.5 and the temperature ranging from15° C. to 37° C.

[0016] As nitrogen sources, ammonia gas, aqueous ammonia, ammonium saltsand the like are used. As inorganic ions, magnesium ion, phosphate ion,potassium ion or iron ions and the like are properly used. By culturingthe transformants under these conditions, prepro-transglutaminase islargely produced in the bacterial cells and extracellularly secreted aspro-transglutaminase and then pro-transglutaminase is cleaved in themedium by a protease which is produced and secreted by a Streptomycesbacterium itself under these condition, then mature (active form)transglutaminase is largely accumulated in the medium.

[0017] Transglutaminases produced and secreted according to the presentinvention can be purified from the medium after culturing, depending ontheir properties, using the methods well known to those skilled in theart. Transglutaminase may be purified, for example, by using knownappropriate techniques such as ammonium sulfate precipitation or ethanolprecipitation, as well as ion-exchange column chromatography,isoelectric focusing or gel filtration or the combination thereof, afterremoving cells, for example, by centrifugation.

EXAMPLES Example 1 Acquisition of Transglutaminase Gene from S.cinnamoneum IFO 12852

[0018] The sequence of transglutaminase from S. cinnamoneum CBS683.68has been already determined (Biochimie., 80, 313-319 (1998)). The primeraccording to SEQ ID NO:5 and SEQ ID NO:6 are synthesized based on thissequence, and the region encoding mature transglutaminase was amplifiedby PCR from chromosomal DNA of S. cinnamoneum IFO 12852 preparedaccording to the method of Saito and Miura (Biochem., biophys., Act.,72, 619 (1963)). PCR was conducted using Pyrobest DNA polymerase (TAKARACo.) under the condition according to its protocol.

[0019] SEQ ID NO:5 5′-TCCGATGACCGGGAAACTCCTCCCGCCGAG-3′

[0020] SEQ ID NO:6 5′-CGGCCAGCCTTGCTCCACCTTGGCGGGGGC-3′

[0021] [Free text in the sequence listing]

[0022] SEQ ID NO:5 :PCR primer for amplifying transglutaminase gene fromS. cinnamoneum

[0023] SEQ ID NO:6 :PCR primer for amplifying transglutaminase gene fromS. cinnamoneum

[0024] The amplified 960 bp DNA fragment are then reacted with[α-³²P]dCTP using Random Primer DNA Labeling Kit Ver.2 (Takara Co.),according to the protocol attached to the kit, to generate the DNAprobe. Using the generated probe and the chromosomal DNA from S.cinnamoneum IFO 12852, Southern blot hybridization was conductedaccording to the conventional procedures such as those described inMolecular Cloning 2nd Edition (J. Sambrook, E. F. Fritsch and T.Maniatis, Cold Spring Harbor Laboratory Press, p9.31 (1989)), whichconfirmed that transglutaminase gene was present in the 3.5 kb fragmentwhich excised by BamHI restriction enzyme. The 3.5 kb BamHI digestedfragment from S. cinnamoneum IFO 12852 chromosomal DNA was recovered byagarose electrophoresis using EASYTRP Ver. 2 (Takara), inserted intoBamHI site of pUC18, and introduced into competent Escherichia coliJM109 cells (Takara) to generate a library. Using the previouslyprepared DNA probe, the library was screened by colony hybridizationaccording to the conventional procedures such as those described inMolecular Cloning 2nd Edition (J. Sambrook, E. F. Fritsch and T.Maniatis, Cold Spring Harbor Laboratory Press, p9.31 (1989)) to obtainthe strain harboring the plasmid where the fragment of transglutaminasegene was cloned. The plasmid was recovered from the strain, which wasdesignated as pB3.5.

[0025] The sequencing of the cloned fragment in pB3.5 confirmed that thetransglutaminase gene from S. cinnamoneum IFO 12852 had almost identicalnucleotide sequence to the transglutaminase gene from S. cinnamoneumCBS683.68. The transglutaminase gene was inserted such that the gene wastranscribed in the direction from EcoRI to HindIII site within themulti-cloning site of pUC18. The sequencing was performed usingDi-terminator Cycle Sequencing Kit (PE Applied Biosystems) and DNASequencer 373A (PE Applied Biosystems).

[0026] The determined nucleotide sequence and the amino acid sequenceencoded by the nucleotide sequence are shown in SEQ ID NO:1 and SEQ IDNO:2, respectively. It was assumed that from amino acid at position 1 toposition 32 in the amino acid sequence is the sequence for pre-part,from amino acid position 33 to position 86 is the sequence for pro-partand from amino acid at position 87 to position 416 is the sequence formature transglutaminase.

Example 2 Construction of the Expression Plasmid for TransglutaminaseGene

[0027] 1) Acquisition of plasmid vector (plJ702)

[0028] plJ702 was prepared according to [J. Bacteriol., 162, 406-412(1985); J. Bacteriol., 169, 1929-1937 (1987)]. More specifically,Streptomyces lividans 3131 (ATCC 35287)(J. Gen. Microbiol., 129,2703-2714 (1983)) obtained by transforming Streptomyces lividans 66 withplJ702 was cultured under the following medium condition at 30° C. for 2days. [YEME medium + 0.5% Glycine + 50 μg/ml thiostrepton] 0.3% YeastExtract 0.5% Peptone 0.3% Malto Extract 0.1% Magnesium chloride 1.0%Glucose 34.0%  Sucrose 0.5% Glycine 0.1% 50 mg/ml thiostrepton solution(Sigma: dimethylsulfoxide solution)(pH 7.0)

[0029] 200 ml of the broth cultured under above condition wascentrifuged (12,000 g, 4° C., 10 min.), washed with 50 mM Tris-HCl(pH(8.0)—5 mM EDTA—50 mM NaCl, and the resulting bacterial cells weresuspended in 10 ml of Tris-HCl (pH8.0)—10 mM EDTA—25% Sucrose (TE-Sucrose). 2 ml of TE-Sucrose containing 30 mg/ml of Lysozyme (Sigma)and 4 ml of 0.25M EDTA was added, the mixture was incubated at 37° C.for 30 minutes, then 2 ml of 20% SDS was added. 5 ml of 5M NaCl wasfurther added, gently mixed, and then, it was incubated overnight. Aftercentrifugation (100,000 g, 4° C., 40 min. ), 30% polyethylene glycol6000 was added to the resulting supernatant at the final concentrationof 10% and the mixture was incubated at 0° C. for 4.5 hours. Then themixture was centrifuged (900 g, 40° C., 5 min.) and the resultingprecipitation was dissolved in 10 mM Tris-HCl (pH 8.0)—1 mM EDTA—50 mMNaCl. To the mixture 1.2 ml of the solution prepared to contain 16.8 gof cesium chloride and 10 mg/ml of ethidium bromide in 10 mMTris-HCl(pH8.0)—1 mM EDTA (referred to as “TE”, hereinafter) was added,the residue was remove by centrifugation (1,300 g, room temperature, 15min.), and then the mixture was centrifuged (230,000 g, 200° C., 12hours). After centrifugation, the plasmid layer was drawn and isolatedunder UV lamp, the solution was repeatedly extracted with TE-saturatedbuthanol 3 times to remove ethidium bromide. The solution was dialyzedagainst TE at 4° C. overnight, extracted once with TE-saturated phenoland twice with chloroform/isoamyl alcohol, and then the aqueous layerwas recovered. Then, 1/10 volume of 3M sodium acetate (pH5.2) solutionand 2 volume of ethanol were added to the solution, and the mixture wasallowed to stand at −80° C. for 30 minutes. The precipitation wasrecovered by centrifugation (12,0000 g, 4° C., 15 minutes), washed with70% ethanol, dried and dissolved in 200 μl of TE. About 10 μg of theplasmid was obtained.

[0030] 2) Construction of the expression plasmid

[0031] A shuttle vector was firstly constructed which can replicate bothin actinomycetes (Streptomyces) host and E.coli host. Multicopy plasmidplJ702 for actinomycetes was digested with SacI and PstI to preparelarge 5.1 kb mel (tyrosine kinase gene) fragment from which the promoterregion was deleted. pOSΔB-Ap1 (about 7.9 kb)(Appl. Environ, Microbiol.,60, 3566-3572 (1994)) into which the fusion gene of protease inhibitorSSI (Streptomyces subtilisin inhibitor) gene and microbial peptide(apidaecin) gene was digested with HindIII and PstI to prepare thefragment of about 2 kb. Multicopy plasmid pUC18 (Takara) for Escherichiacoli was digested with EcoRI, blunted with T4 DNA polymerase (Takara),and self ligated. Plamids which could not be digested with EcoRI wereselected and the plasmids were digested with SacI and HindIII to preparethe 2.7 kb fragment. Then, shuttle vector pUJS (about 9.8 kb) wasconstructed by the ternary ligation of the 5.1 kb SacI-PstI fragmentfrom plJ702, the 2 kb HindIII-PstI fragment from pOSΔB-Ap1 and the 2.7kb SacI-HindIII fragment from pUC1 8.

[0032] pUJS was digested with HindIII and EcoRI to recover the large 8.6kb fragment. pB3.5 (about 6.2 kb) containing transglutaminase gene fromS. cinnamoneum IFO 12852 which was cloned according to (1) was digestedwith HindIII and EcoRI and the 3.5 kb HindIII-EcoRI fragment wasrecovered. The 3.5 kb HindIII-EcoRI fragment was inserted into HindIIIand EcoRI site of pUJS to construct pUJ-MTG (about 12.1 kb). The abovedescribed construction procedures are shown in FIG. 1.

[0033]E. coli AJ13669 obtained by transforming E.coli with pUJ-MTG wasdeposited in the National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology (1-3, Higashi 1 chomeTsukuba-shi Ibaraki-ken 305-8566, JAPAN) (The microorganism had beendeposited as FERM P-17602 on Oct. 14, 1999 and was transferred to theinternational deposit based on the Budapest Treaty as FERM BP-7287 onAug. 28, 2000).

Example 3 Transformation of S. lividans TK24

[0034]S. lividans TK24 is the strain derived from S. lividans 66, whichis mounted a streptomycin resistance (GENETIC MANIPULATION OFSTREPTOMYCES, A LABORATORY MANYAL: D. A. Hopwood et al., p266, 1985, Thejohn Innes Foundation Norwich). This strain was provided from D. A.Hopwood (John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K.) andis obtainable from D. A. Hopwood's laboratory. S. lividans Tk24 wastreated to make protoplasts and transformed according to the method of[JP-Kokai No. 3-251182; Hunter, I. S., “DNA Cloning” A PracticalApproach 2, Glover, D. M.(Ed.) IRL Press (1985), GENETIC MANIPULATION OFSTREPTOMYCES, A LABORATORY MANUAL: D. A. Hopwood et al., p104, 1985, TheJohn Innes Foundation Norwich]. More specifically, S. lividans wascultured in YEME medium+0.5% Glycine at 30° C. for two(2) days. 200 mlof broth was centrifuged(1,300 g, room temperature, 10 min.) and theresulting bacterial cells were suspended in 72 ml of 0.35M sucrose.

[0035] The suspension was then centrifuged (1,300 g, room temperature,10 min.), incubated at 30° C. for 2.5 hours, filtrated with absorbentcotton to remove residues. The resulting filtrate was centrifuged (1,300g, room temperature, 10 min.) and the precipitant was washed twice with25 ml of P-buffer and suspended in 1 ml of P-buffer to prepare aprotoplast suspension.

[0036] [P-buffer] TES[N-Tris(hydroxymethyl)methyl- 5.73 g 2-aminoethanesulphonic acid] Sucrose 103 g Magnesium chloride 2.03 g Potassiumsulfate 0.5 g Calcium chloride 3.68 g Trace elements solution 2 ml/L (pH7.4)

[0037] 1 ml of 1% potassium phosphate solution per 100 ml of P-buffer,which had been separately prepared, was added to P-buffer immediatelybefore use.

[0038] [Trace elements solution]

[0039] It contains the followings per 1L of the solution: Zinc chloride40 mg Ferric chloride 200 mg  Cupric chloride 10 mg Manganese chloride10 mg Tetra sodium borate 10 mg ammonium molybdate 10 mg

[0040] The transformation of S. lividans TK24 protoplast suspension withpUJ-MTG (about 12.1 kb), transglutaminase gene expressing plasmid, wasconducted as follows. DNA solution (0.2 μg/μl) 20 μl Protoplastsuspension of S. lividans TK24 100 μl 0.35 M Sucrose 20 μl P-buffercontaining 20% polyethylene glycol 1000 1.5 ml

[0041] was gently mixed and allowed to stand for 2 minutes at the roomtemperature.

[0042] The mixture was centrifuged (1,700 g, room temperature, 10 min.)and the pellet was collected, repeatedly washed twice with P-buffer,suspended in 1 ml of P-buffer and spreaded on the following R-2 agarplates.

[0043] [R-2 Agar plate]

[0044] 1) R-2/A 1) R-2/A Potassium sulfate 0.5 g/l Magnesium chloride20.2 g/l Calcium chloride 5.9 g/l Glucose 20.0 g/l Proline 6.0 g/lCasamino acid 0.2 g/l Trace elements solution 4 ml Agar 44.0 g/l 2)R-2/B TES 11.5 g/l Yeast Extract 10.0 g/l Sucrose 203 g/l (pH7.4)

[0045] 3) 1% KH₂PO₄

[0046] 1), 2) and 3) were separately prepared. R-2/A and R-2/B weremixed on the preparation of plates and 1 ml of 1 % KH₂PO₄ solution perfinal volume of 200ml of the mixture was added. R-2 agar plates wherethe transformants were plated were incubated at 30° C. for 18 hours. 1ml of P-buffer containing 200 μg/ml of thiostrepton was poured on thepate to cover the entire surface of the agar and the plates were furtherincubated at 30° C. for 7 days to obtain colonies. Plasmids wereprepared from the obtained colonies to confirm that the intended plasmidwas introduced.

Example 4 The Expression of Transglutaminase and Secretory Production

[0047] The transformant pUJ-MTGIS.lividans TK24 was cultured in 4 ml ofTripton-Soya Broth (DIFCO) liquid medium containing 10 μg/ml ofthiostrepton at 30° C. for 3 days. 1 ml of the culture was seeded into100 ml of the same liquid medium in 500 ml Sakaguchi flask and culturedat 30° C. for 2 weeks. The samples were taken sequentially from theculture and 10 μl of the broth supernatant was subjected to SDS-PAGE andthen to Western blot analysis using the anti-transglutaminase antibodydescribed in JP-Kokai No.6-046855 according to the general method suchas described in Molecular Cloning 2nd Edition (J. Sambrook, E. F.Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p18.60(1989)). As a result, the secretory production (about 40-50 mg/l) oftransglutaminase with additional pro-structural part was found tillabout day 7 to 10 of culturing and then the yield of transglutaminasehaving almost the same molecular weight as that of maturetransglutaminase, which can be the result of processing ofpro-transglutaminase, increased on further culturing. At about week 2 inculturing, about 40-50 mg/l of mature transglutaminase was accumulated.

[0048] Using the supernatant of broth at the phase where the amount ofsecretory production of transglutaminase with additional pro-structuralpart (pro-transglutaminase) was large, SDS-PAGE and semi-dry blotting toPVDF membrane were conducted ( Analysis of Protein Structure for GeneCloning, Tokyo Kagaku Dojin (1993)). After blotting, the PVDF membranewas stained with Coomassie brilliant blue, de-stained and dried in air.The portion corresponding to pro-transglutaminase was excised andanalyzed for the N-terminal amino acid sequence by the protein sequencer(Model 476A, Perkin-Elmer). The result confirmed the 10 amino acidssequence (Gly-Asp-Gly-Glu-Glu-Lys-Gly-Ser-Tyr-Ala-, SEQ ID NO:7) ofpro-transglutaminase. This amino acid sequence differed from thesequence of pro-region indicated in Biochimie., 80, 313-319 (1998), butwas identical to the amino acid sequence determined in Example 1 (SEQ IDNO:2).

[0049] According to the present invention, a large amount oftransglutaminase can be obtained in the broth by directing Streptomycesbacteria to produce transglutaminase. Since transglutaminase accumulatedin the broth according to the present invention is maturetransglutaminase cleaved by proteases which are produced by theStreptomyces bacteria themselves, mature transglutaminase can be easilyrecovered from the broth on a large scale.

1 7 1 1461 DNA Streptoverticillium cinnamoneum CDS (151)..(1398) 1cggcggcagc cctccttgcc gccggcgcag cgacgcagga cggcgcggcc aaggccctga 60gcggcagctc gtcgcaaacc cctccatcgc gtcgtgctct cacatgccct cgtttcacga 120ggcttcacca caagggagtt attgatttcc atg cac aaa cgt cgg aga ctt ctc 174 MetHis Lys Arg Arg Arg Leu Leu 1 5 gcc ttc gcc act gtg ggt gcg gtc ata tgcacc gca gga ttc aca cct 222 Ala Phe Ala Thr Val Gly Ala Val Ile Cys ThrAla Gly Phe Thr Pro 10 15 20 tcg gtc agc cag gcc gcc agc agt ggc gat ggggaa gag aag ggg tcc 270 Ser Val Ser Gln Ala Ala Ser Ser Gly Asp Gly GluGlu Lys Gly Ser 25 30 35 40 tac gcc gaa acg cac ggc ctg acg gcg gat gacgtc gag agc atc aac 318 Tyr Ala Glu Thr His Gly Leu Thr Ala Asp Asp ValGlu Ser Ile Asn 45 50 55 gca ctg aac gaa aga gct ctg act ctg ggc caa cctggc aag cct ccg 366 Ala Leu Asn Glu Arg Ala Leu Thr Leu Gly Gln Pro GlyLys Pro Pro 60 65 70 aag gaa tta cct ccg agc gcc agc gcg ccc tcc cgg gccccc tcc gat 414 Lys Glu Leu Pro Pro Ser Ala Ser Ala Pro Ser Arg Ala ProSer Asp 75 80 85 gac cgg gaa act cct ccc gcc gag ccg ctc gac agg atg cctgag gcg 462 Asp Arg Glu Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro GluAla 90 95 100 tac cgg gcc tac gga ggc agg gcc act acg gtc gtc aac aactac ata 510 Tyr Arg Ala Tyr Gly Gly Arg Ala Thr Thr Val Val Asn Asn TyrIle 105 110 115 120 cgc aag tgg cag cag gtc tac agt cac cgc gac gga aagaaa cag caa 558 Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Lys LysGln Gln 125 130 135 atg acc gaa gag cag cga gaa aag ctg tcc tac ggt tgcgtt ggc gtc 606 Met Thr Glu Glu Gln Arg Glu Lys Leu Ser Tyr Gly Cys ValGly Val 140 145 150 acc tgg gtc aac tcg ggc ccc tac ccg acg aac aga ttggcg ttc gcg 654 Thr Trp Val Asn Ser Gly Pro Tyr Pro Thr Asn Arg Leu AlaPhe Ala 155 160 165 tcc ttc gac gag aac aag tac aag aac gac ctg aag aacacc agc ccc 702 Ser Phe Asp Glu Asn Lys Tyr Lys Asn Asp Leu Lys Asn ThrSer Pro 170 175 180 cga ccc gat gaa acg cgg gcg gag ttc gag ggt cgc atcgcc aag ggc 750 Arg Pro Asp Glu Thr Arg Ala Glu Phe Glu Gly Arg Ile AlaLys Gly 185 190 195 200 agt ttc gac gag ggg aag ggt ttc aag cgg gcg cgtgat gtg gcg tcc 798 Ser Phe Asp Glu Gly Lys Gly Phe Lys Arg Ala Arg AspVal Ala Ser 205 210 215 gtc atg aac aag gcc ctg gaa aat gcc cac gac gagggg act tac atc 846 Val Met Asn Lys Ala Leu Glu Asn Ala His Asp Glu GlyThr Tyr Ile 220 225 230 aac aac ctc aag acg gag ctc acg aac aac aat gacgct ctg ctc cgc 894 Asn Asn Leu Lys Thr Glu Leu Thr Asn Asn Asn Asp AlaLeu Leu Arg 235 240 245 gag gac agc cgc tcg aac ttc tac tcg gcg ctg aggaac aca ccg tcc 942 Glu Asp Ser Arg Ser Asn Phe Tyr Ser Ala Leu Arg AsnThr Pro Ser 250 255 260 ttc aag gaa agg gac ggc ggc aac tac gac ccg tccaag atg aag gcg 990 Phe Lys Glu Arg Asp Gly Gly Asn Tyr Asp Pro Ser LysMet Lys Ala 265 270 275 280 gtg atc tac tcg aag cac ttc tgg agc ggg caggac cag cgg ggc tcc 1038 Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln AspGln Arg Gly Ser 285 290 295 tcc gac aag agg aag tac ggc gac ccg gaa gccttc cgc ccc gac cag 1086 Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala PheArg Pro Asp Gln 300 305 310 ggt acc ggc ctg gtc gac atg tcg aag gac agaagc att ccg cgc agt 1134 Gly Thr Gly Leu Val Asp Met Ser Lys Asp Arg SerIle Pro Arg Ser 315 320 325 ccg gcc aag ccc ggc gaa ggt tgg gtc aat ttcgac tac ggt tgg ttc 1182 Pro Ala Lys Pro Gly Glu Gly Trp Val Asn Phe AspTyr Gly Trp Phe 330 335 340 ggg gct caa aca gaa gcg gat gcc gac aaa accaca tgg acc cac ggc 1230 Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr ThrTrp Thr His Gly 345 350 355 360 gac cac tac cac gcg ccc aat agc gac ctgggc ccc atg cac gta cac 1278 Asp His Tyr His Ala Pro Asn Ser Asp Leu GlyPro Met His Val His 365 370 375 gag agc aag ttc cgg aag tgg tct gcc gggtac gcg gac ttc gac cgc 1326 Glu Ser Lys Phe Arg Lys Trp Ser Ala Gly TyrAla Asp Phe Asp Arg 380 385 390 gga gcc tac gtg atc acg ttc ata ccc aagagc tgg aac acc gcc ccc 1374 Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys SerTrp Asn Thr Ala Pro 395 400 405 gcc aag gtg gag caa ggc tgg ccgtgacaggctg gtactacgac ctctgctgat 1428 Ala Lys Val Glu Gln Gly Trp Pro410 415 ttctgcccgg tcagtccacg cctctcgacg cga 1461 2 416 PRTStreptoverticillium cinnamoneum 2 Met His Lys Arg Arg Arg Leu Leu AlaPhe Ala Thr Val Gly Ala Val 1 5 10 15 Ile Cys Thr Ala Gly Phe Thr ProSer Val Ser Gln Ala Ala Ser Ser 20 25 30 Gly Asp Gly Glu Glu Lys Gly SerTyr Ala Glu Thr His Gly Leu Thr 35 40 45 Ala Asp Asp Val Glu Ser Ile AsnAla Leu Asn Glu Arg Ala Leu Thr 50 55 60 Leu Gly Gln Pro Gly Lys Pro ProLys Glu Leu Pro Pro Ser Ala Ser 65 70 75 80 Ala Pro Ser Arg Ala Pro SerAsp Asp Arg Glu Thr Pro Pro Ala Glu 85 90 95 Pro Leu Asp Arg Met Pro GluAla Tyr Arg Ala Tyr Gly Gly Arg Ala 100 105 110 Thr Thr Val Val Asn AsnTyr Ile Arg Lys Trp Gln Gln Val Tyr Ser 115 120 125 His Arg Asp Gly LysLys Gln Gln Met Thr Glu Glu Gln Arg Glu Lys 130 135 140 Leu Ser Tyr GlyCys Val Gly Val Thr Trp Val Asn Ser Gly Pro Tyr 145 150 155 160 Pro ThrAsn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asn Lys Tyr Lys 165 170 175 AsnAsp Leu Lys Asn Thr Ser Pro Arg Pro Asp Glu Thr Arg Ala Glu 180 185 190Phe Glu Gly Arg Ile Ala Lys Gly Ser Phe Asp Glu Gly Lys Gly Phe 195 200205 Lys Arg Ala Arg Asp Val Ala Ser Val Met Asn Lys Ala Leu Glu Asn 210215 220 Ala His Asp Glu Gly Thr Tyr Ile Asn Asn Leu Lys Thr Glu Leu Thr225 230 235 240 Asn Asn Asn Asp Ala Leu Leu Arg Glu Asp Ser Arg Ser AsnPhe Tyr 245 250 255 Ser Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg AspGly Gly Asn 260 265 270 Tyr Asp Pro Ser Lys Met Lys Ala Val Ile Tyr SerLys His Phe Trp 275 280 285 Ser Gly Gln Asp Gln Arg Gly Ser Ser Asp LysArg Lys Tyr Gly Asp 290 295 300 Pro Glu Ala Phe Arg Pro Asp Gln Gly ThrGly Leu Val Asp Met Ser 305 310 315 320 Lys Asp Arg Ser Ile Pro Arg SerPro Ala Lys Pro Gly Glu Gly Trp 325 330 335 Val Asn Phe Asp Tyr Gly TrpPhe Gly Ala Gln Thr Glu Ala Asp Ala 340 345 350 Asp Lys Thr Thr Trp ThrHis Gly Asp His Tyr His Ala Pro Asn Ser 355 360 365 Asp Leu Gly Pro MetHis Val His Glu Ser Lys Phe Arg Lys Trp Ser 370 375 380 Ala Gly Tyr AlaAsp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile 385 390 395 400 Pro LysSer Trp Asn Thr Ala Pro Ala Lys Val Glu Gln Gly Trp Pro 405 410 415 31809 DNA Streptoverticillium mobaraense CDS (578)..(1798) 3 gtcgacgcgggccgggaggg ggtgcggcgg cgcccttcgg ctgtgtggac gaagcgtcgg 60 gtcggaggggcggccggata tcgtccttgg ggcggggtgg ccggaattgc cgccatggtg 120 ttgccggggaatcgacccga agacatgatc acttctcgta tccacccgat cacgtatccg 180 ggagtcgagaagtgttacgc cgtgcccctg tccgcgtcct cacccctgtc gccgtgacag 240 cgacccgcgttcttccactc gcacggacgg ccccacagga cctttcggcc cgggctcgcc 300 ccgccgcctcggtgacggcc tccgaataac gcggccgccg gggcctcggc cggttgaccg 360 atccgggtcacgcgccccgc cgggcgggcg gccacgtccg gtctcgcccc gcccgacatc 420 ggctgcgactgccttcgctc gcacttcttc ccgcctcccg gccgcgtttt tccgccgccg 480 aaggtgcggcgacgcgtacc gaatccccct tcatcgcgac gtgcttccgc acggccgcgt 540 tcaacgatgttccacgacaa aggagttgca ggtttcc atg cgc ata cgc cgg aga 595 Met Arg IleArg Arg Arg 1 5 gct ctc gtc ttc gcc act atg agt gcg gtg tta tgc acc gccgga ttc 643 Ala Leu Val Phe Ala Thr Met Ser Ala Val Leu Cys Thr Ala GlyPhe 10 15 20 atg ccg tcg gcc ggc gag gcc gcc gcc gac aat ggc gcg ggg gaagag 691 Met Pro Ser Ala Gly Glu Ala Ala Ala Asp Asn Gly Ala Gly Glu Glu25 30 35 acg aag tcc tac gcc gaa acc tac cgc ctc acg gcg gat gac gtc gcg739 Thr Lys Ser Tyr Ala Glu Thr Tyr Arg Leu Thr Ala Asp Asp Val Ala 4045 50 aac atc aac gcg ctc aac gaa agc gct ccg gcc gct tcg agc gcc ggc787 Asn Ile Asn Ala Leu Asn Glu Ser Ala Pro Ala Ala Ser Ser Ala Gly 5560 65 70 ccg tcg ttc cgg gcc ccc gac tcc gac gac agg gtc acc cct ccc gcc835 Pro Ser Phe Arg Ala Pro Asp Ser Asp Asp Arg Val Thr Pro Pro Ala 7580 85 gag ccg ctc gac agg atg ccc gac ccg tac cgt ccc tcg tac ggc agg883 Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg 9095 100 gcc gag acg gtc gtc aac aac tac ata cgc aag tgg cag cag gtc tac931 Ala Glu Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr 105110 115 agc cac cgc gac ggc agg aag cag cag atg acc gag gag cag cgg gag979 Ser His Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu 120125 130 tgg ctg tcc tac ggc tgc gtc ggt gtc acc tgg gtc aat tcg ggt cag1027 Trp Leu Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln 135140 145 150 tac ccg acg aac aga ctg gcc ttc gcg tcc ttc gac gag gac aggttc 1075 Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe155 160 165 aag aac gag ctg aag aac ggc agg ccc cgg tcc ggc gag acg cgggcg 1123 Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala170 175 180 gag ttc gag ggc cgc gtc gcg aag gag agc ttc gac gag gag aagggc 1171 Glu Phe Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly185 190 195 ttc cag cgg gcg cgt gag gtg gcg tcc gtc atg aac agg gcc ctggag 1219 Phe Gln Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu200 205 210 aac gcc cac gac gag agc gct tac ctc gac aac ctc aag aag gaactg 1267 Asn Ala His Asp Glu Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu215 220 225 230 gcg aac ggc aac gac gcc ctg cgc aac gag gac gcc cgt tccccg ttc 1315 Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser ProPhe 235 240 245 tac tcg gcg ctg cgg aac acg ccg tcc ttc aag gag cgg aacgga ggc 1363 Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg Asn GlyGly 250 255 260 aat cac gac ccg tcc agg atg aag gcc gtc atc tac tcg aagcac ttc 1411 Asn His Asp Pro Ser Arg Met Lys Ala Val Ile Tyr Ser Lys HisPhe 265 270 275 tgg agc ggc cag gac cgg tcg agt tcg gcc gac aag agg aagtac ggc 1459 Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg Lys TyrGly 280 285 290 gac ccg gac gcc ttc cgc ccc gcc ccg ggc acc ggc ctg gtcgac atg 1507 Asp Pro Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu Val AspMet 295 300 305 310 tcg agg gac agg aac att ccg cgc agc ccc acc agc cccggt gag gga 1555 Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro GlyGlu Gly 315 320 325 ttc gtc aat ttc gac tac ggc tgg ttc ggc gcc cag acggaa gcg gac 1603 Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr GluAla Asp 330 335 340 gcc gac aag acc gtc tgg acc cac gga aat cac tat cacgcg ccc aat 1651 Ala Asp Lys Thr Val Trp Thr His Gly Asn His Tyr His AlaPro Asn 345 350 355 ggc agc ctg ggt gcc atg cat gtc tac gag agc aag ttccgc aac tgg 1699 Gly Ser Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe ArgAsn Trp 360 365 370 tcc gag ggt tac tcg gac ttc gac cgc gga gcc tat gtgatc acc ttc 1747 Ser Glu Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val IleThr Phe 375 380 385 390 atc ccc aag agc tgg aac acc gcc ccc gac aag gtaaag cag ggc tgg 1795 Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp Lys Val LysGln Gly Trp 395 400 405 ccg tgatgtgagc g 1809 Pro 4 407 PRTStreptoverticillium mobaraense 4 Met Arg Ile Arg Arg Arg Ala Leu Val PheAla Thr Met Ser Ala Val 1 5 10 15 Leu Cys Thr Ala Gly Phe Met Pro SerAla Gly Glu Ala Ala Ala Asp 20 25 30 Asn Gly Ala Gly Glu Glu Thr Lys SerTyr Ala Glu Thr Tyr Arg Leu 35 40 45 Thr Ala Asp Asp Val Ala Asn Ile AsnAla Leu Asn Glu Ser Ala Pro 50 55 60 Ala Ala Ser Ser Ala Gly Pro Ser PheArg Ala Pro Asp Ser Asp Asp 65 70 75 80 Arg Val Thr Pro Pro Ala Glu ProLeu Asp Arg Met Pro Asp Pro Tyr 85 90 95 Arg Pro Ser Tyr Gly Arg Ala GluThr Val Val Asn Asn Tyr Ile Arg 100 105 110 Lys Trp Gln Gln Val Tyr SerHis Arg Asp Gly Arg Lys Gln Gln Met 115 120 125 Thr Glu Glu Gln Arg GluTrp Leu Ser Tyr Gly Cys Val Gly Val Thr 130 135 140 Trp Val Asn Ser GlyGln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser 145 150 155 160 Phe Asp GluAsp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg 165 170 175 Ser GlyGlu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser 180 185 190 PheAsp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val 195 200 205Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp 210 215220 Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu 225230 235 240 Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro SerPhe 245 250 255 Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met LysAla Val 260 265 270 Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg SerSer Ser Ala 275 280 285 Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe ArgPro Ala Pro Gly 290 295 300 Thr Gly Leu Val Asp Met Ser Arg Asp Arg AsnIle Pro Arg Ser Pro 305 310 315 320 Thr Ser Pro Gly Glu Gly Phe Val AsnPhe Asp Tyr Gly Trp Phe Gly 325 330 335 Ala Gln Thr Glu Ala Asp Ala AspLys Thr Val Trp Thr His Gly Asn 340 345 350 His Tyr His Ala Pro Asn GlySer Leu Gly Ala Met His Val Tyr Glu 355 360 365 Ser Lys Phe Arg Asn TrpSer Glu Gly Tyr Ser Asp Phe Asp Arg Gly 370 375 380 Ala Tyr Val Ile ThrPhe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp 385 390 395 400 Lys Val LysGln Gly Trp Pro 405 5 30 DNA Artificial Sequence synthetic DNA primerfor amplification of S. cinnamoneum transglutaminase gene 5 tccgatgaccgggaaactcc tcccgccgag 30 6 30 DNA Artificial Sequence synthetic DNAprimer for amplification of S. cinnamoneum transglutaminase gene 6cggccagcct tgctccacct tggcgggggc 30 7 10 PRT Streptoverticilliumcinnamoneum 7 Gly Asp Gly Glu Glu Lys Gly Ser Tyr Ala 1 5 10

1. A bacterium belonging to Streptomyces, wherein a gene constructcontaining a transglutaminase gene from actinomycetes and a promotersequence controlling the transglutaminase gene is introduced into thebacterium.
 2. The bacterium belonging to Streptomyces according to claim1, wherein the actinomycetes is Streptoverticillium cinnamoneum.
 3. Thebacterium belonging to Streptomyces according to claim 1 or 2, whereinthe bacterium belonging to Streptomyces is Streptomyces lividans.
 4. Amethod of producing a pro-transglutaminase derived from actinomycetes,comprising the steps of culturing the bacterium belonging toStreptomyces according to any one of claims 1 to 3, and allowing thebacterium to secrete the pro-transglutaminase into a culture medium. 5.A method of producing a mature transglutaminase derived fromactinomycetes, comprising the steps of culturing the bacterium belongingto Streptomyces according to any one of claims 1 to 3, allowing thebacterium to secrete a pro-transglutaminase derived from actinomycetesinto a culture medium, cleaving the pro-structural part of thepro-transglutaminase by a protease derived from said bacterium belongingto Streptomyces and recovering the mature transglutaminase.
 6. Themethod according to claim 4, wherein the pro-transglutaminase has theamino acid sequence from Glycine residue at position 33 to Prolineresidue at position 416 in the sequence of SEQ ID NO:2.
 7. The methodaccording to claim 5, wherein the mature transglutaminase has the aminoacid sequence from Serine residue at position 87 to Proline residue atposition 416 in the sequence of SEQ ID NO:2.